Solid phase based biomedical assays, especially ELISAs (enzyme-linked immunosorbent assays) are used very commonly in the field of clinical diagnostics and biological research. They are widely used analysing methods due to the ease and rapidity of the assay procedures. There are diverse materials used as supporting solid phase, e.g., (microtiter-) plates or particles (beads) made of polymers like polystyrene, polypropylene and other substances. Well-established tests on the market range from serological diagnosis of severe diseases (e.g., HIV, Hepatitis, TSE, Malaria, Tuberculosis) to routine controls in clinical chemistry (e.g., cholesterol, insulin, drug/pregnancy tests).
Many reagents or sample components in biological assays tend to adhere to the solid phase or to already immobilised reagents on the surface in an unintended manner. As such binding does not represent a part of the very specific ligand/receptor (e.g., antigen-antibody) recognition process, it is generally referred to as non-specific binding (NSB). NSB events (NSBs), especially of detection antibodies, potentially lead to false-positive signals, as they may occur even if one component of the intended recognition setup is missing. Such false-positives are inherent to the system and appear as an even background signal or simply “noise”. On the other hand, NSBs may disturb the recognition process between ligand and receptor (e.g., antigen and antibody). While false-positives lower the specificity of an assay, a disturbance of the intended detection reactions by NSBs may also lead to a reduction of the signal-to-noise ratio, thereby generating false-negatives which in turn reduce the sensitivity of an assay.
In total, NSBs are a huge problem, because in many cases they are a major factor for inferior signal-to-noise ratios (S/N) of solid phase bioassays. Although the sensitivity and the detection limit of an assay often can be improved by using higher amounts of reagents, this approach is not advantageous in general, as it is costly, and it often causes higher NSB rates as well, thereby limiting the advantage of a higher sensitivity by a lower specificity. Additionally, the amount of sample provided is limited in many situations. Here in particular the reduction of NSB is of utmost importance in order to improve an assay.
Many different possibilities for the reduction of NSB are known in the state of the art. They range from modifying antibodies, changing the incubation conditions such as pH value, (pre-)purification of sample material or enrichment of the analyte of interest to treating the sample with serum or heat. These methods are often tedious, very problem-specific and their success tends to vary with assay conditions and the reagents used, so that they need to be newly developed for each experiment and do not always exhibit the desired effect.
A very common procedure for the prevention of NSB is the saturation of the solid phase surface with a so-called blocking reagent. In general, solid phase surfaces used for bioanalytical assays are designed in such a way that adsorption of biological materials is very facile. In a first step of the assay, referred to as coating step, one assay component onto which further assay components shall bind in subsequent assay steps is adsorbed onto the surface of the solid phase. This coating component usually does not cover the entire surface of the solid phase. Consequently, an additional assay step, referred to as blocking step, is required, where remaining free space on the solid phase surface is covered by a so-called blocking reagent. If blocking is omitted all biological components present in the following incubation steps may become adsorbed on remaining free spots of the surface, thereby causing the main part of NSB.
By treating the solid phase with a blocking reagent after the coating step, the surface ought to be saturated and this way protected against adsorption of further material. The effectiveness of the blocking procedure may vary greatly and depends largely on the type of blocking reagent applied in the assay.
To date, many different blocking reagents are used and are commercially available. The most common ones include reagents from biological sources, such as animal sera, gelatine, skimmed milk, treated or non-treated proteins and protein fractions like bovine serum albumin (BSA), casein or casein hydrolysate, but also detergents and polymers like Tween20 or Poly(vinylpyrrolidone) (PVP) (Studentsov et al. (2002). Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus. J. Clin. Microbiol. 40:1755-1760).
Blocking reagents provide a sufficient solution for many NSB problems, but the reagents available to date have drawbacks. Firstly, most blocking reagents are derived from biological sources and, thus, are not only heterogeneous, lot-to-lot variable and decomposable, but may also be subject to import and export restrictions due to potential biohazards of certain materials of biological origin. They also tend to cross-react and may even inhibit important recognition processes like streptavidin-biotin binding. Problems with cross-reactivity have led to the development of reagents like fish sera, which show less cross-reactivity with mammalian reagents. Some blocking reagents derived from biological material, e.g., skimmed milk, are known to possess very good NSB reducing abilities, but they may also decrease the specificity of the assay by covering or replacing the coating material.
Synthetic blocking reagents, e.g., Tween20, do not share these disadvantages of proteinaceous materials, but they are not sufficient in reducing NSB and could therefore not establish a solid market share.
In conclusion, blocking reagents are normally obligatory in a solid phase based immunoassay, but it is not easy to determine which reagent is the most appropriate one. Many experimental assays are still not used in routine applications, because the available blocking reagents are not effective enough and the sensitivity of detection is insufficient.
Sufficing specificity is equally difficult to attain. Often high specificity is only achieved by cutting back on sensitivity and/or by accepting some of the above mentioned drawbacks (e.g., lot-to-lot variability) of blocking reagents from biological sources).